Molecular Survey of Legionella pneumophila in Dental Unit Waterlines at Najaf Dental Clinics-Iraq

Research Article

J Dent & Oral Disord. 2017; 3(1): 1049.

Molecular Survey of Legionella pneumophila in Dental Unit Waterlines at Najaf Dental Clinics-Iraq

Taher AA*, Alsehlawi ZS and Al-Yasiri IK

Department of Basic Science, College of Dentistry, Kufa University, Iraq

*Corresponding author: Abbas AY Taher, Department of Maxillofacial Surgery, College of Dentistry, Kufa University, Iraq

Received: January 12, 2017; Accepted: February 09, 2017; Published: February 16, 2017


Aims: The present study was intended to determine the occurrence of Legionella spp in Dental Unit Water Lines (DUWL) among public and private dental clinics in Najaf, Iraq.

Methods and Results: Ninety-four water samples were randomly selected from various parts of the dental unit. Standardized 50ml water samples were taken from the DUWL. The processed sample was cultured on Based Charcoal Yeast Extract Medium (BCYE) plates. After incubation, the macroscopic and microscopic examinations were done to describe the bacterial morphology. Polymerase Chain Reaction (PCR) was used to examine the presence of Legionella pneumophila in tested samples. The presence of Legionella spp. was involved in 34 (36.1%) DUWL samples and detected in 28 (70.0%) of 40 bottled water samples using BCYE agar. The PCR experiment for detection of L. pneumophila revealed that 10/34 (29.4%) isolates were positive for the 16 sr RNA gene. Most of them 9/28 (32.1%) were isolated from dental unit bottles followed by one isolate that was identified in syringe water.

Conclusions: It can be concluded that L. pneumophila is moderately distributed in the DUWL of Najaf dental clinics and considered a contaminating agent that may lead to a serious risk of patient infection.

Significance and impact of study: Statistical analysis showed that the culture method had excellent correlation with PCR results (P>0.0001).

Keywords: Legiomella spp; Dental water lines; PCR assay; 16 sr RNA gene; Dental clinics


Legionella spp. are now known to be free-living organisms surviving as natural components of freshwater ecosystems [1]. Although some phenotypic characteristics of Legionella spp. are small Gram-negative fastidious and microaerophillic rods [2]. They are encapsulated and non-spore forming. Legionella can survive in varied water conditions, in temperatures ranging from 0-63oC and pH ranging from 5.0-8.5 [3]. Biofilm formation occurs on the inner surface of the water lines as a result of water stagnation [4,5]. Hence, the water system of the hospitals with endemic Legionella may be the main source of nosocomial legionellosis responsible for mild upper respiratory tract infections or pneumonia following inhalation of contaminated water droplets from a variety of water sources [6]. Approximately 3% to 8% of all community-acquired pneumonias are caused by Legionella spp. and 85% of those pneumonias are caused by L. pneumophila [7]. The majority of the organisms in the biofilm are harmless environmental species, but some dental units may harbor opportunistic respiratory pathogens such as Legionellae [8]. Several studies have indicated that dentists and dental staff in addition to the patients admitted to the dental clinics have higher rates of respiratory infections than the general public [9]. Thus, contaminated water lines and hand pieces are believed to be at least partially responsible for these higher rates of respiratory disease. The present study intended to determine the proliferation of Legionella spp. in dental water lines at different public and private dental clinics in Najaf, Iraq.

Materials and Methods

Samples collection

During the period from Sept. 2015 to Feb. 2016, a total of 90 dental units of public and private clinics were randomly selected and screened. While, 94 diverse water samples (50 ml) were collected from bottles, Syringes and High-Speed Drills (HSD) for each dental unit, then transported to the advanced laboratory of microbiology in the Basic Science Department and kept at 8oC until used.

Preparation of sample for bacteriologic examination

Acid buffer was used to reduce the number of non-Legionella bacteria in water samples before culture according to the [1]. This buffer consisted of two solutions prepared as follows A: 0.2 M KCl (14.9 g/L in distilled water) and B: 0.2 M HCl (16.7 g/L in distilled water). Mix 18 parts of (A) with 1 part of (B) dispensed into screwcapped tubes in 1.0 ml volumes and sterilized by autoclaving. Mix 1.0 ml of the vortexed centrifuged water sample into a sterile 15 ml centrifuge tube containing 1.0 ml of sterile acid buffer. The acidified suspension mixture was incubated for 15 minutes at room temperature.

Culture and examination for Legionella

Volume 0.1 ml of the suspension was placed onto BCYE plates (previously prepared included supplement, Himedia, India) and spread with a sterile glass rod. Plates were incubated at 35oC in a candle jar with an atmosphere of 2.5% CO2 using a gas generation kit, Oxoid, UK. [1]. All cultures were examined after 3 to 4 days of incubation. Negative plates were re-incubated for seven days and then reexamined for Legionella spp. colonies. Plates were discarded after the seventh day. Macroscopic examination was conducted using a dissecting microscope to detect bacterial colonies resembling Legionella. Microscopic examination was conducted; Gram stain smear was prepared to detect stain, shape and arrangement of bacteria. Bacterial isolates with convex colonies and round edges were suspected. The center of the colony is usually bright white in color with a textured appearance that has been described as “cut-glasslike.” Cells are small Gram-negative rods according to [10,11].

DNA extraction and PCR assay

DNA was extracted from bacterial cells performed as described by Wizard Minipreps DNA kit (Promiga, USA). The DNA was used as a template for PCR amplification for the 16s rRNA gene of L. pneumophila (386 bp). PCR amplification was carried out in a gradient thermal cycler (T professional Basic Biometra, Germany) using primers (IDT, USA) according to [12]. JFP/F (5-AGGGGTTGATAGGTTAAGAGC-3’) and JFP/R (5’-CCAACAGCTAGTTGACATCG-3’). For amplification, an initial denaturation was conducted at 95oC for 20 minutes followed by 38 cycles of denaturation at 94oC for 45 seconds, annealing at 57oC for 45 seconds and elongation for 45 seconds at 72oC. The final extension step was 1 hour at 72oC. The PCR products were analyzed by agarose gel electrophoresis using 1.5% agarose. Bands were visualized with UV-transilluminator (UVP, USA) after being stained with ethidium bromide.

Statistical analysis

The (chi-square) test was used to evaluate the positivity rate of isolation and molecular identification of the Legionella spp. in DUWL samples by the proposed methods.


This is the first study in Iraq related to molecular detection of Legionella spp. in DUWL. Water samples were collected from various parts of dental units and investigated for the presence of Legionella spp. by culture and confirmed using PCR. Overall, there were 94 acid buffer-treated samples; 34 (36.1%) of them had heavy Legionella spp. growth on BCYE media. (Table 1) shows the number of detected Legionella spp. using BCYE and the source of the tested samples. Twenty-eight were isolated from bottled water. However, 4 were identified in syringe and 2 in HSD. Subsequently, to detect L. penumophila at the molecular level, the 16Sr RNA gene was amplified using PCR (Figure 1). Of the 34 total culture positive isolates, 10 (29.4%) were PCR positive for 16Sr RNA. Samples of bottled water mostly harbored L. pneumophila 9 (32.1%), followed by one isolate (25.0%) identified in syringe water (Table 2). This study revealed that no L. pneumophila was detected in HSD water samples by PCR. Statistical analysis showed that the culture method had excellent correlation with PCR results (P>0.0001).